We have used mice in which the gene for cytosolic phospholipase A₂ (cPLA₂) has been disrupted to demonstrate the absolute requirement for cPLA₂ in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA₂ prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D₂ generation. These defects occurred with two distinct stimuli, stem cell factor and IgE/antigen, which were, however, sufficient for signal transduction defined by exocytosis of β-hexosaminidase. Whereas cPLA₂ is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD₂ generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD₂.